Promote Aggregate Formation Research Articles

Flagellar motility and the mucus environment influence aggregation mediated antibiotic tolerance of Pseudomonas aeruginosa in chronic lung infection.

Antibiotic recalcitrance of chronic Pseudomonas aeruginosa infections in muco-obstructive airway diseases is a primary driver of mortality. Mechanisms that drive antibiotic tolerance are poorly understood. We investigated motility phenotypes related to P. aeruginosa adaptation and antibiotic tolerance in the diseased mucus environment. Loss of flagellar motility drives antibiotic tolerance by promoting aggregate formation. Regulation of flagellar motility appears to be a key step in aggregate formation as the inability to turn off flagellin expression resulted in poor aggregate formation and increased antibiotic susceptibility. These results deepen our understanding of the formation of antibiotic tolerant aggregates within the MADs airway and opens novel avenues and targets for treatment of chronic P. aeruginosa infections.

6432 Enriching Testicular Organoid Aggregates to Promote Germ Cell Homing

Abstract Disclosure: A. Patel: None. O.O. Printy: None. C. Joswiak: None. M.M. Laronda: None. The long-term effects of life-saving chemotherapy treatments, such as an increased risk of infertility, are of concern to the growing number of childhood cancer survivors. De novo testicular morphogenesis seeks to generate testicular organoids that are structurally and functionally similar to the native testis. These organoids serve as a tool for investigating patient-specific models for restoring spermatogenesis for prepubertal cancer patients. Our lab has previously developed testicular organoids containing tubule-like structures (TLS) resembling the native seminiferous tubules as assessed by immunohistochemistry of testicular cell-type markers for Leydig cells (HSD3B2), peritubular myoid cells (ACTA1), Sertoli cells (SOX9), germ cells (DDX4), and spermatogonial stem cells (SSCs) (SALL4). Internal architecture demonstrated semi-continuous ring-like structures composed of ACTA1-positive cells encapsulating a population of SOX9-positive cells with HSD3B2-positive cells clustering near the periphery of the organoids. However, organoids lacked native germ cell populations. We hypothesized that murine testicular organoids would merge to form elongated TLS, within which exogenous SSCs would home to existing Sertoli cell niches, when cultured within structures designed from the appropriate dimensions and materials. Organoids were generated from testicular tissue isolated from 5 days-post-partum (dpp) CD1 mice using cell-seeding densities and microenvironmental conditions previously established by our lab. To promote aggregate formation, 48-hour-old organoids were transferred to trough-shaped structures formed from 2% agarose using 3D printed silicone molds. Separately, SSCs were isolated from 5 dpp tdTomato+ mice via magnetic-activated cell sorting of THY1+ cells. Organoids and aggregates were enriched with SSCs by three methods: (1) enrichment of cell suspension during organoid formation, (2) enrichment of culture during aggregate formation, and (3) injection of SSCs into aggregate using 20 µm glass pipette. Organoids transferred to agarose troughs on day 2 of culture merged to form aggregates by day 5 and continued to display dynamic architecture, shortening in length and increasing in width over time. Aggregates were more elongated in shape when cultured in narrow troughs less than 700µm in width. Observations thus far indicate that attempts to inject aggregates resulted in penetrance and inclusion of the SSC suspension; additionally, tdTomato+ cells were visualized within injected aggregates on day 17 of culture. Research is ongoing to define where exogenous SSCs take up residence, how long they survive, and if they proliferate over time within the testicular aggregates. This research expands upon foundational work establishing testicular organoids as an approach to in vitro spermatogenesis with clinical applications in fertility restoration. Presentation: 6/2/2024

6432 Enriching Testicular Organoid Aggregates to Promote Germ Cell Homing

Abstract Disclosure: A. Patel: None. O.O. Printy: None. C. Joswiak: None. M.M. Laronda: None. The long-term effects of life-saving chemotherapy treatments, such as an increased risk of infertility, are of concern to the growing number of childhood cancer survivors. De novo testicular morphogenesis seeks to generate testicular organoids that are structurally and functionally similar to the native testis. These organoids serve as a tool for investigating patient-specific models for restoring spermatogenesis for prepubertal cancer patients. Our lab has previously developed testicular organoids containing tubule-like structures (TLS) resembling the native seminiferous tubules as assessed by immunohistochemistry of testicular cell-type markers for Leydig cells (HSD3B2), peritubular myoid cells (ACTA1), Sertoli cells (SOX9), germ cells (DDX4), and spermatogonial stem cells (SSCs) (SALL4). Internal architecture demonstrated semi-continuous ring-like structures composed of ACTA1-positive cells encapsulating a population of SOX9-positive cells with HSD3B2-positive cells clustering near the periphery of the organoids. However, organoids lacked native germ cell populations. We hypothesized that murine testicular organoids would merge to form elongated TLS, within which exogenous SSCs would home to existing Sertoli cell niches, when cultured within structures designed from the appropriate dimensions and materials. Organoids were generated from testicular tissue isolated from 5 days-post-partum (dpp) CD1 mice using cell-seeding densities and microenvironmental conditions previously established by our lab. To promote aggregate formation, 48-hour-old organoids were transferred to trough-shaped structures formed from 2% agarose using 3D printed silicone molds. Separately, SSCs were isolated from 5 dpp tdTomato+ mice via magnetic-activated cell sorting of THY1+ cells. Organoids and aggregates were enriched with SSCs by three methods: (1) enrichment of cell suspension during organoid formation, (2) enrichment of culture during aggregate formation, and (3) injection of SSCs into aggregate using 20 µm glass pipette. Organoids transferred to agarose troughs on day 2 of culture merged to form aggregates by day 5 and continued to display dynamic architecture, shortening in length and increasing in width over time. Aggregates were more elongated in shape when cultured in narrow troughs less than 700µm in width. Observations thus far indicate that attempts to inject aggregates resulted in penetrance and inclusion of the SSC suspension; additionally, tdTomato+ cells were visualized within injected aggregates on day 17 of culture. Research is ongoing to define where exogenous SSCs take up residence, how long they survive, and if they proliferate over time within the testicular aggregates. This research expands upon foundational work establishing testicular organoids as an approach to in vitro spermatogenesis with clinical applications in fertility restoration. Presentation: 6/2/2024

Reducing the Sodium Adsorption Ratio Improves the Soil Aggregates and Organic Matter in Brackish-Water-Irrigated Cotton Fields

The assessment of soil health relies on key parameters such as soil aggregates and organic matter content. Therefore, examining the impact of irrigation water ion composition and variations in salinity on soil aggregates and organic matter is imperative, which is key to developing a theoretical basis for the sustainable utilization of saline water resources, particularly in extremely arid regions. This experiment was conducted to investigate the impact of different irrigation water salinity treatments (T3: 3 g/L, T5: 5 g/L, and T7: 7 g/L) on the root zone soil of cotton fields. Each salinity treatment included three variations of the sodium adsorption ratio (SAR) at S10: 10 (mmol/L)1/2, S15: 15 (mmol/L)1/2, and S20: 20 (mmol/L)1/2. Local freshwater irrigation served as the control, resulting in a total of 10 treatments. Our findings show that the soil Ca2+ and Mg2+ content increased with higher irrigation water salinity but decreased with increasing irrigation water SAR. The relative macroaggregate stability and the content of water-stable macroaggregates and soil organic matter (SOM) decreased as the irrigation water salinity and SAR increased. In comparison to T3S20, T5S10 did not improve the soil Na+ content but significantly increased the soil Ca2+ content by 147.76%, while the water-stable aggregate and SOM saw a notable increase of 7.66% and 9.86%, respectively. Reducing the SAR in brackish water lessens its negative impact on soil aggregates in cotton fields. This is primarily because Ca2+ counteracts the dispersive effect of high Na+ concentrations and promotes aggregate formation. Irrigation water with a salinity of 3 g/L and an SAR of 10 (mmol/L)1/2 positively affected the stabilization of soil aggregates and organic matter.

Assessing the formation and stability of paddy soil aggregate driven by organic carbon and Fe/Al oxides in rice straw cyclic utilization strategies: Insight from a six-year field trial

Soil organic carbon (SOC) and iron/aluminum (Fe/Al) oxides are key cementing agents in driving soil aggregate formation, yet their direct effects and interactions on aggregate under long-term rice straw cyclic utilization (LSCU) in cold regions are still unclear. We compared chemical fertilizer (CF) with LSCU strategy: rice-straw (RS), biochar (RB), and biochar-based fertilizer (BF). We showed that the increase of macroaggregate (2–0.25 mm) is associated with SOC, dissolved organic carbon (DOC), humin carbon (HUC), amorphous and organic complexed Fe/Al oxides (Feo, Fep, Alo, Alp), and in each size of the aggregate, there exists an interaction between SOC (fractions) and Fe/Al oxides. Furthermore, aggregate stability was determined by Feo, Fep, and Alo. LSCU enhances macroaggregate and aggregate stability by increasing SOC and Fe/Al oxides in the bulk soil and aggregates, but there are differences among LSCU. In all treatments, RS had more DOC, fulvic acid carbon (FAC), humic acid carbon (HAC) and Fep; while RB had more SOC, HUC, free Fe/Al oxides (Fed, Ald), Feo, Alp; and BF had more Alo in bulk soil. Over the years, RS increased the DOC, FAC and HAC, whereas RB enhanced the stable SOC fractions (HUC) and promoted high reactive Fe/Al oxides formation (Feo, Fep, Alo), and BF increased DOC, Feo, Fep and Alo. Moreover, RB increases the direct pathway of SOC and Fe/Al oxides to aggregate, promoting aggregate formation. Our study provides new perspective on the mechanisms and promising practice for improving rice straw utilization efficiently, paddy soil fertility and productivity sustainably in cold regions.

Straw returning combined with controlled-release nitrogen fertilizer affected organic carbon storage and crop yield by changing humic acid composition and aggregate distribution

Soil organic carbon (SOC) sequestration effectively reduces global CO2 emissions while ensuring crop production and sustainable agricultural development. Evaluating the effects of straw returning combined with controlled-release nitrogen (N) fertilizer on soil organic carbon (SOC) sequestration is necessary. We conducted a long-term (2013–2018) insitu field trial with six treatments: straw combined with no N and two N fertilizer types [no N fertilizer (CKS), conventional N fertilizer (BBFS) and controlled-release N fertilizer (CRFS)] and their corresponding no N and two N fertilizer treatments (CK, BBF, and CRF), each in three replicates. Overall, straw returning combined with N fertilizer (BBFS and CRFS) improves carbon storage and crop yield by changing aggregate distribution, with the effect of CRFS being more substantial. We further found CRFS treatment significantly increased SOC storage in small macro-aggregates (0.25–2 mm) by 9.2% and 28.4%, respectively, compared to BBFS and CKS treatments, which is mainly due to CRFS treatment increasing the proportion of small macro-aggregates (0.25–2 mm) by 8.1% and 20.1%, respectively, in contrast with BBFS and CKS treatments. Compared to BBFS and CKS treatments, CRFS treatment increased the fluorescence intensity of hydrophobic compounds in humic acid (HA) by 45.0% and 107.6%, respectively. Similarly, in small macro-aggregates (0.2–2.5 mm), CRFS treatment significantly increased the fluorescence intensity of hydrophobic compounds in HA. Lastly, structural equation modeling showed that straw returning treatments promote aggregate formation and carbon sequestration by increasing the fluorescence intensity of hydrophobic compounds in HA, ultimately increasing crop yield. Therefore, CRFS treatment is theoretically an effective method to promote SOC storage and increase crop yield and is a viable practice for sustainable agriculture.

Desmin mutations impact the autophagy flux in C2C12 cell in mutation-specific manner.

Desmin is the main intermediate filament of striated and smooth muscle cells andplays a crucial role in maintaining the stability of muscle fiber during contraction and relaxation cycles. Being a component of Z-disk area, desmin integrates autophagic pathways, and the disturbance of Z-disk proteins' structure negatively affects chaperone-assisted selective autophagy (CASA). In the present study, we focused on alteration of autophagy flux in myoblasts expressing various Des mutations. We applied Western blotting, immunocytochemistry, RNA sequencing, and shRNA approach to demonstrate that DesS12F, DesA357P, DesL345P, DesL370P, and DesD399Y mutations. Mutation-specific effect on autophagy flux being most severe in aggregate-prone Des mutations such as DesL345P, DesL370P, and DesD399Y. RNA sequencing data confirmed the most prominent effect of these mutations on expression profile and, in particular, on autophagy-related genes. To verify CASA contribution to desmin aggregate formation, we suppressed CASA by knocking down Bag3 and demonstrated that it promoted aggregate formation and lead to downregulation of Vdac2 and Vps4a and upregulation of Lamp, Pink1, and Prkn. In conclusion, Des mutations showed a mutation-specific effect on autophagy flux in C2C12 cells with either a predominant impact on autophagosome maturation or on degradation and recycling processes. Aggregate-prone desmin mutations lead to the activation of basal autophagy level while suppressing the CASA pathway byknocking down Bag3 can promote desmin aggregate formation.

A mechanistic model of cross-bridge migration in RBC aggregation and disaggregation.

Red blood cells (RBCs) clump together under low flow conditions in a process called RBC aggregation, which can alter RBC perfusion in a microvascular network. As elevated RBC aggregation is commonly associated with cardiovascular and inflammatory diseases, a better understanding of aggregation is essential. Unlike RBC aggregation in polymer solutions which can be well explained by polymer depletion theory, plasma-mediated RBC aggregation has features that best match explanations with cross-bridging mechanisms. Previous studies have demonstrated the dominant role of fibrinogen (Fg) in promoting aggregate formation and recent cell-force spectroscopy (CFS) experiments on interacting RBC doublets in plasma have reported an inverse relationship between disaggregation force and the adhesive contact area between RBCs. This has led investigators to revisit the hypothesis of inter-RBC cross-bridging which involves cross-bridge migration under interfacial tension during the forced disaggregation of RBC aggregates. In this study, we developed the cross-bridge migration model (CBMM) in plasma that mechanistically represents the migrating cross-bridge hypothesis. Transport of mobile Fg cross-bridges (mFg) was calculated using a convection-diffusion transport equation with our novel introduction of convective cross-bridge drift that arises due to intercellular friction. By parametrically transforming the diffusivity of mFg in the CBMM, we were able to match experimental observations of both RBC doublet formation kinematics and RBC doublet disaggregation forces under optical tweezers tension. We found that non-specific cross-bridging promotes spontaneous growth of adhesion area between RBC doublets whereas specific cross-bridging tends to prevent adhesion area growth. Our CBMM was also able to correlate Fg concentration shifts from healthy population blood plasma to SLE (lupus) condition blood plasma with the observed increase in doublet disaggregation forces for the RBC doublets in SLE plasma.

Understanding Importance of Soil Testing and Significance of SOC on Crop Productivity

Healthy soils are important to grow healthy crops, raising healthy animals, and supporting a healthy human population through nutritionally balanced diets. As an indicator for soil health, SOC is important for its contributions to food production, mitigation and adaptation to climate change, and the achievement of the Sustainable Development Goals (SDGs). It supports multiple soil functions determining soil physical, chemical and biological quality parameters contributing to the productive capacity of soil. It improves soil structural stability by promoting aggregate formation which, together with porosity, ensures sufficient aeration and water infiltration to support plant growth. It is one of the factors that help in increasing water holding capacity, improves cation exchange capacity to hold huge quantities of positively charged nutrients such as calcium, magnesium and potassium until the plant needs them.

Tyrosine 136 phosphorylation of \u03b1-synuclein aggregates in the Lewy body dementia brain: involvement of serine 129 phosphorylation by casein kinase 2

Serine 129 (S129) phosphorylation of α-synuclein (αSyn) is a central feature of Lewy body (LB) disease pathology. Although the neighboring tyrosine residues Y125, Y133, and Y136 are also phosphorylation sites, little is known regarding potential roles of phosphorylation cross-talk between these sites and its involvement in the pathogenesis of LB disease. Here, we found that αSyn aggregates are predominantly phosphorylated at Y136 in the Lewy body dementia brain, which is mediated by unexpected kinase activity of Casein kinase 2 (CK2). Aggregate formation with S129 and Y136 phosphorylation of recombinant αSyn (r-αSyn) were induced by CK2 but abolished by replacement of S129 with alanine (S129A) in vitro. Mutation of Y136 to alanine (Y136A) promoted aggregate formation and S129 phosphorylation of r-αSyn by CK2 in vitro. Introduction of Y136A r-αSyn oligomers into cultured cells exhibited increased levels of aggregates with S129 phosphorylation compared to wild-type r-αSyn oligomers. In addition, aggregate formation with S129 phosphorylation induced by introduction of wild-type r-αSyn oligomers was significantly attenuated by CK2 inhibition, which resulted in an unexpected increase in Y136 phosphorylation in cultured cells. Our findings suggest the involvement of CK2-related αSyn Y136 phosphorylation in the pathogenesis of LB disease and its potential as a therapeutic target.

The formation of large macroaggregates induces soil organic carbon sequestration in short-term cropland restoration in a typical karst area

Cropland restoration induces litter and root inputs and promotes the development of biological soil crusts (BSCs), which may promote aggregate formation and soil organic carbon (SOC) sequestration. However, litter, roots and BSCs have not been simultaneously considered when assessing soil aggregate and aggregate-associated SOC fraction responses to cropland restoration in subtropical areas. Here, we measured particulate organic carbon (POC) and mineral-associated organic carbon (MOC) in bulk soils and soil aggregates after 15 years of cropland restoration. Soil samples of cropland (CL) and four cropland restoration types (plantation forest [PF], forage grassland [FG], mixed plantation of forest and forage grassland [FF], and abandoned natural grassland [NG]) from depths of 0–30 cm were collected. Cropland restoration significantly increased SOC and POC in bulk soil at the 0–5 cm depth. However, only in FG did SOC significantly increase at depths of 5–15 cm, and POC significantly increased at depths of 5–30 cm. The large macroaggregate (5–10 mm and 2–5 mm) proportions increased significantly at the 0–15 cm depth after cropland restoration, and FG, FF and NG also increased the 5–10 mm aggregate proportions at the 15–30 cm depth. The SOC sequestration in bulk soil with cropland restoration was attributed to increases in the aggregate-associated organic carbon (AAOC) pool in large macroaggregates, which was mainly attributed to the increased aggregate amount rather than the increased AAOC concentration in large macroaggregates. Our results also indicated that an increase in aggregate-associated particulate organic carbon (AAPOC) led to an increase in AAOC. Variation partitioning indicated that the formation of large macroaggregates was controlled by the litter-moss-root interactive effect in this karst area. FG could be a better short-term cropland restoration strategy, increasing large macroaggregates in deeper soil layers better than the other vegetation types and promoting soil carbon sequestration in deeper soil layers.

Texture Analysis of Dried Droplets for the Quality Control of Medicines.

The quality control of medicines guarantees the effectiveness of treatments for diseases. We explore the use of texture analysis of patterns in dried droplets as a tool to readily detect both impurities and changes in drug concentration. Four types of medicines associated with different routes of administration were analyzed: Methotrexate, Ciprofloxacin, Clonazepam, and Budesonide. We use NaCl and a hot substrate at 63 C to promote aggregate formation and to reduce droplet drying time. Depending on the medicine, optical microscopy reveals different complex aggregates such as circular to oval splatters, fern-like islands, crown shapes, crown needle-like and bump-like patterns as well as dendritic branched and star-like crystals. We use some physical features of the stains (as the stain diameter and superficial area) and gray level co-occurrence matrix (GLCM) to characterize patterns of dried droplets. Finally, we show that structural analysis of stains can achieve 95% accuracy in identifying medicines with 30% water dilution, while it achieves 99% accuracy in detecting drugs with 10% other substances.

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model.

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a large proportion of the infecting bacteria form biofilms within airway sputum. Pa biofilms isolated from CF sputum have been shown to grow in small, dense aggregates of ~10-1,000 cells that are spatially organized and exhibit clinically relevant phenotypes such as antimicrobial tolerance. One of the biggest challenges to studying how Pa aggregates respond to the changing sputum environment is the lack of nutritionally relevant and robust systems that promote aggregate formation. Using a synthetic CF sputum medium (SCFM2), the life history of Pa aggregates can be observed using confocal laser scanning microscopy (CLSM) and image analysis at the resolution of a single cell. This in vitro system allows the observation of thousands of aggregates of varying size in real time, three dimensions, and at the micron scale. At the individual and population levels, having the ability to group aggregates by phenotype and position facilitates the observation of aggregates at different developmental stages and their response to changes in the microenvironment, such as antibiotic treatment, to be differentiated with precision.

In vitro Exopolysaccharide Production of Some Soil Bacteria Isolated from Soil Affected by Salt

In this study, production of exopolysaccharide (EPS) from soil bacteria isolated from the salinity soil and the effects of agitation, some carbon sources such as glucose, fructose, xylose, rhamnose and NaCl levels the on production of polysaccharide playing a role in soil aggregation were studied. Maximum EPS production for Acinetobacter sp., Pseudomonas fluorescens and Sphingomonas paucimobilis was obtained 72 h of incubation and maximum EPS production for Bacillus simplex was obtained at 96 h. Maximum EPS production was obtained at 150 rpm for all bacteria studied. Glucose gave the best result for all of bacteria. Bacillus simplex produced higher EPS and viscosity than the other bacteria. NaCl levels affected both the growth and exopolysaccharide production of the bacteria. By promoting aggregate formation, EPS produced bacteria such as S.paucimobilis, B.simplex, Acinetobacter sp. and P.fluorescens may have created favorable growth environments.

High glycine content in TDP-43: a potential culprit in limbic-predominant age-related TDP-43 encephalopathy.

Numerous risk factors for heart disease or dementia harbor over 10% valine plus glycine content. Interestingly, TDP-43 contains 6.0% valine and 13.3% glycine, and the buildup of this protein in the brains of patients with limbic-predominant age-related TDP-43 encephalopathy has dire consequences. The two γ-methyl groups in valine enable hyperconjugation, which enhances the van der Waals interaction between its side group and the carbonyl carbon. This extends the C=O bond length, and this weakened C=O bond augments the secondary chemical bonding of the carbonyl oxygen atom to cations. This, in turn, promotes the formation and buildup of insoluble and rigid salts such as calcium oxalate, which is postulated to be a major cause of heart disease. Similarly, the long C=O bond length in glycine results in a weakened C=O bond with an enhanced affinity toward cations and the formation of insoluble salts. Further, several prion proteins possess a high glycine content of approximately 20%. The insoluble calcium salts produced may promote aggregate formation via secondary chemical bonding between calcium and glycine, as well as between calcium and valine. Chemical and biochemical insights will help us to better understand the etiology of disorders linked to protein aggregates.

Removal of harmful algal blooms in freshwater by buoyant-bead flotation using chitosan-coated fly ash cenospheres.

Harmful algal blooms (HABs) are a growing problem worldwide, damaging human and ecosystem health. In this study, a novel buoyant-bead flotation (BBF) method using chitosan-coated fly ash cenospheres (CFACs) was developed to remove HABs in freshwater. To achieve a high removal efficiency of harmful algae (Chlorella vulgaris, Scenedesmus quadricauda, and Microcystis aeruginosa), this study investigated the effects of chitosan/fly ash ratios in CFAC composite, CFAC concentration, flotation time, and pH values on the microalgae removal. The optimized ratio of CFACs is 0.1:12, and the optimized CFAC concentration is 0.3-0.7gL-1. However, the lower or higher ratios (0.1:4, 0.1:8, 0.1:16) result in microalgae reaching a zero-point charge too late or early, which failed to effectively remove HABs with an appropriate coal fly ash dosage. An optimized removal efficiency of 98.50% for Microcystis aeruginosa was reached at pH of 6.0. The optimized efficiency of Scenedesmus quadricauda and Chlorella vulgaris was 99.37% and 91.63%, respectively, at pH of 8.0. At neutral pH conditions, the surface charge of microalgae cells and CFACs are different, promoting aggregate formation. When CFACs were used to remove microalgae, aggregate size significantly influenced removal efficiency. Meanwhile, at the optimized pH and concentration, the removal efficiency of all three algal species exceeded 90.00% in 5min. The study highlights an efficient and inexpensive method for removing HABs and obtains the optimized operational conditions.

Correction: Investigation of synovial fluid induced Staphylococcus aureus aggregate development and its impact on surface attachment and biofilm formation.

[This corrects the article DOI: 10.1371/journal.pone.0231791.].

Investigation of synovial fluid induced Staphylococcus aureus aggregate development and its impact on surface attachment and biofilm formation.

Periprosthetic joint infections (PJIs) are a devastating complication that occurs in 2% of patients following joint replacement. These infections are costly and difficult to treat, often requiring multiple corrective surgeries and prolonged antimicrobial treatments. The Gram-positive bacterium Staphylococcus aureus is one of the most common causes of PJIs, and it is often resistant to a number of commonly used antimicrobials. This tolerance can be partially attributed to the ability of S. aureus to form biofilms. Biofilms associated with the surface of indwelling medical devices have been observed on components removed during chronic infection, however, the development and localization of biofilms during PJIs remains unclear. Prior studies have demonstrated that synovial fluid, in the joint cavity, promotes the development of bacterial aggregates with many biofilm-like properties, including antibiotic resistance. We anticipate these aggregates have an important role in biofilm formation and antibiotic tolerance during PJIs. Therefore, we sought to determine specifically how synovial fluid promotes aggregate formation and the impact of this process on surface attachment. Using flow cytometry and microscopy, we quantified the aggregation of various clinical S. aureus strains following exposure to purified synovial fluid components. We determined that fibrinogen and fibronectin promoted bacterial aggregation, while cell free DNA, serum albumin, and hyaluronic acid had minimal effect. To determine how synovial fluid mediated aggregation affects surface attachment, we utilized microscopy to measure bacterial attachment. Surprisingly, we found that synovial fluid significantly impeded bacterial surface attachment to a variety of materials. We conclude from this study that fibrinogen and fibronectin in synovial fluid have a crucial role in promoting bacterial aggregation and inhibiting surface adhesion during PJI. Collectively, we propose that synovial fluid may have conflicting protective roles for the host by preventing adhesion to surfaces, but by promoting bacterial aggregation is also contributing to the development of antibiotic tolerance.

Ube2v1 Positively Regulates Protein Aggregation by Modulating Ubiquitin Proteasome System Performance Partially Through K63 Ubiquitination.

Compromised protein quality control can result in proteotoxic intracellular protein aggregates in the heart, leading to cardiac disease and heart failure. Defining the participants and understanding the underlying mechanisms of cardiac protein aggregation is critical for seeking therapeutic targets. We identified Ube2v1 (ubiquitin-conjugating enzyme E2 variant 1) in a genome-wide screen designed to identify novel effectors of the aggregation process. However, its role in the cardiomyocyte is undefined. To assess whether Ube2v1 regulates the protein aggregation caused by cardiomyocyte expression of a mutant αB crystallin (CryABR120G) and identify how Ube2v1 exerts its effect. Neonatal rat ventricular cardiomyocytes were infected with adenoviruses expressing either wild-type CryAB (CryABWT) or CryABR120G. Subsequently, loss- and gain-of-function experiments were performed. Ube2v1 knockdown decreased aggregate accumulation caused by CryABR120G expression. Overexpressing Ube2v1 promoted aggregate formation in CryABWT and CryABR120G-expressing neonatal rat ventricular cardiomyocytes. Ubiquitin proteasome system performance was analyzed using a ubiquitin proteasome system reporter protein. Ube2v1 knockdown improved ubiquitin proteasome system performance and promoted the degradation of insoluble ubiquitinated proteins in CryABR120G cardiomyocytes but did not alter autophagic flux. Lys (K) 63-linked ubiquitination modulated by Ube2v1 expression enhanced protein aggregation and contributed to Ube2v1's function in regulating protein aggregate formation. Knocking out Ube2v1 exclusively in cardiomyocytes by using AAV9 (adeno-associated virus 9) to deliver multiplexed single guide RNAs against Ube2v1 in cardiac-specific Cas9 mice alleviated CryABR120G-induced protein aggregation, improved cardiac function, and prolonged lifespan in vivo. Ube2v1 plays an important role in protein aggregate formation, partially by enhancing K63 ubiquitination during a proteotoxic stimulus. Inhibition of Ube2v1 decreases CryABR120G-induced aggregate formation through enhanced ubiquitin proteasome system performance rather than autophagy and may provide a novel therapeutic target to treat cardiac proteinopathies.

Molecular characterization of soil organic carbon in water-stable aggregate fractions during the early pedogenesis from parent material of Mollisols

The aims of this study were to investigate how short-term agricultural practices varying in vegetation cover, tillage, and organic amendments affected the chemistry of aggregate-associated C and particulate organic matter (POM) after 8-year soil restoration from parent material (PM) of a Mollisol. The research allowed indicating the effects of perennialization and organic amendments on the chemical compositions of soil organic carbon (SOC) in both aggregates and POM in short-term and thereof helped to unveil the mechanisms of C sequestration and stabilization. The field surface soils (0–20 cm) under 8-year natural perennials and an arable soil with organic amendments were collected. Soil aggregate fractionation method was applied to obtain three aggregate-sized fractions: macroaggregate (> 250 μm), microaggregate (53–250 μm), and silt-clay unit ( 250 μm), microaggregate (53–250 μm), and silt-clay units within macroaggregate, the fine POM (fPOM, 53–250 μm), and silt-clay units within microaggregate were further separated. In all, 10 subgroups of aggregates were obtained to determine organic carbon content and chemical structure using 13C-NMR spectroscopy technique. The PM for the establishment of the experiment was also used. The chemical composition of SOC differed among aggregate sizes, representing different organic compositions with different decomposition stages. Compared with PM, soil restoration under natural perennial and organic amendments increased SOC in both POM and aggregate fractions; higher proportion of POM with larger proportion of plant-derived O-alkyl C was observed in arable soil than in perennials. Meanwhile, the silt-clay units, sequestrating the most of the organic C, contained larger proportions of alkyl C, aromatic C, and carbonyl C, probably due to the microbial-derived by-products. The data partially supported our hypothesis that continuous organic amendments to PM likely promoted aggregate formation and subsequently affected the chemical composition of C therein. Perennialization and organic amendments increased not only the total SOC stocks in bulk soils and aggregate fractions but also changed the SOM chemistry by varying quantity and quality of plant residues. The silt-clay units contributed largely to the stock and stability of SOC during the soil development stage from PM of a Mollisol. The main mechanisms of plant-derived C sequestrated and SOC accrued were controlled probably not only by the physiochemical protection of soil aggregates but also by the microbial C sequestration capacity in silt-clay fraction at the initial stage of soil formation.